Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

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Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells

Journal of Biological Standardization ◽

10.1016/0092-1157(74)90016-x ◽

1974 ◽

Vol 2 (3) ◽

pp. 203-209 ◽

Cited By ~ 64

Author(s):

Kikuko Miyamura ◽

Etsuko Tajiri ◽

A. Ito ◽

R. Murata ◽

R. Kono

Keyword(s):

Cell Culture ◽

Diphtheria Toxin ◽

Culture Method ◽

Vero Cells

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Recombinant Staphylococcus Strains as Live Vectors for the Induction of Neutralizing Anti-Diphtheria Toxin Antisera

Infection and Immunity ◽

10.1128/iai.67.10.5007-5011.1999 ◽

1999 ◽

Vol 67 (10) ◽

pp. 5007-5011 ◽

Cited By ~ 9

Author(s):

Cécile Fromen-Romano ◽

Pascal Drevet ◽

Alain Robert ◽

André Ménez ◽

Michel Léonetti

Keyword(s):

Diphtheria Toxin ◽

Expression System ◽

Vero Cells ◽

Heterologous Proteins ◽

Potential Vector ◽

Toxic Protein ◽

Gram Positive Bacteria ◽

Live Bacteria ◽

Sequence Encoding ◽

Bacterial Lysates

ABSTRACT We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosuscan display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE′mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S. xylosus and S. carnosus cell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface of S. xylosus cells whereas it is efficiently exposed on S. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally with S. carnosus displaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, only S. carnosus can display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.

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Towards a Recombinant Vaccine against Diphtheria Toxin

Infection and Immunity ◽

10.1128/iai.66.2.418-423.1998 ◽

1998 ◽

Vol 66 (2) ◽

pp. 418-423 ◽

Cited By ~ 15

Author(s):

Karin Lobeck ◽

Pascal Drevet ◽

Michel Léonetti ◽

Cécile Fromen-Romano ◽

Frédéric Ducancel ◽

...

Keyword(s):

Receptor Binding ◽

Diphtheria Toxin ◽

Protein A ◽

Vero Cells ◽

Binding Domain ◽

Receptor Binding Domain ◽

Surface Receptors ◽

Linear Peptide ◽

Recombinant Fragments

ABSTRACT Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT168–220) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT168–220, and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT168–220, which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT168–220 antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

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